title: Ordered and dynamic assembly of single spliceosomes.
abstract: The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Hoskins
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Gallagher
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Crawford
authors.forename: Eric G
authors.initials: EG
authors.lastname: Anderson
authors.forename: Richard
authors.initials: R
authors.lastname: Wombacher
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ramirez
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
authors.forename: Jeff
authors.initials: J
authors.lastname: Gelles
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Moore
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D005456:Fluorescent Dyes; D007438:Introns; D007700:Kinetics; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; C079877:PRP19 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate